High pressure refolding of monoclonal antibody aggregates

ABSTRACT

Methods for refolding antibodies, particularly monoclonal antibodies, from aggregated and/or denatured preparations by subjecting the antibody preparation to high hydrostatic pressure are provided. Refolded preparations of antibodies produced by the methods described herein are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional PatentApplication No. 60/971,223, filed Sep. 10, 2007. The entire contents ofthat application are hereby incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the high pressure refolding of antibodyaggregates, and in particular monoclonal antibody aggregates.

BACKGROUND OF THE INVENTION

Many proteins are valuable as therapeutic agents. Protein therapeuticsare often produced using recombinant DNA technology, which can enableproduction of higher amounts of protein than can be isolated fromnaturally-occurring sources, and which avoids contamination that oftenoccurs with proteins isolated from naturally-occurring sources.

Proper folding of a protein is essential to the normal functioning ofthe protein. Improperly folded proteins are believed to contribute tothe pathology of several diseases, including Alzheimer's disease, bovinespongiform encephalopathy (BSE, or “mad cow” disease) and humanCreutzfeldt-Jakob disease (CJD), and Parkinson's disease; these diseasesserve to illustrate the importance of proper protein folding.

Proteins of therapeutic value in humans can be expressed in bacteria,yeast, and other microorganisms. While large amounts of proteins can beproduced in such systems, the proteins are often misfolded, and oftenaggregate together in large clumps called inclusion bodies. The proteinscannot be used in the misfolded, aggregated state. Accordingly, methodsof disaggregating and properly refolding such proteins have been thesubject of much investigation.

One method of refolding proteins uses high pressure on solutions ofproteins in order to disaggregate, unfold, and properly refold proteins.Such methods are described in U.S. Pat. No. 6,489,450, U.S. Pat. No.7,064,192, U.S. Patent Application Publication No. 2004/0038333, andInternational Patent Application WO 02/062827. Those disclosuresindicated that certain high-pressure treatments of aggregated proteinsor misfolded proteins resulted in recovery of disaggregated proteinretaining biological activity (i.e., the protein was properly folded, asis required for biological activity) in good yields. U.S. Pat. No.6,489,450, U.S. Pat. No. 7,064,192, U.S. 2004/0038333, and WO 02/062827are incorporated by reference herein in their entireties.

Certain devices have also been developed which are particularly suitablefor refolding of proteins under high pressure; see International PatentApplication Publication No. WO 2007/062174, which is incorporated byreference herein in its entirety.

Several monoclonal antibodies are currently in use as therapeuticagents, for example, Herceptin® (Trastuzumab) (Herceptin® is aregistered trademark of Genentech, Inc., South San Francisco, Calif.,for a monoclonal antibody useful in treating breast cancer) andRemicade® (Infliximab) (Remicade® is a registered trademark of Centocor,Inc., Malvern, Pa., for a monoclonal antibody useful in treatinginflammatory disorders involving the immune system such as rheumatoidarthritis). Unfortunately, some of the most widely used processing stepsfor monoclonal antibody production, such as Protein A/G affinitypurification and/or viral inactivation steps, require use of solutionsat pH levels as low as approximately pH 3.0 during typicalpharmaceutical protein manufacturing (Ejima et al., Proteins, 66:954-62(2007)). Monoclonal antibodies readily aggregate during treatment at pH3.0, possibly due to destabilization of the Fc domain. These aggregatescan be difficult to remove and result in increased production costs.See, Thommes, J. and M. Etzel, Biotechnology Progress 23(1): 42-45(2007) for a discussion of these issues. High pressure refoldingprovides a viable method for alleviating aggregation of monoclonalantibodies induced by manufacturing processes.

The effect of aggregate formation conditions on the pressure-modulatedrefolding yield is currently unknown. An earlier report (St. John, R.J., J. F. Carpenter, et al., Journal of Biological Chemistry 276(50):46856-46863 (2001)) showed that the refolding yields of recombinanthuman growth hormone from two different insoluble aggregates containeddifferent secondary structures and resulted in different refoldingkinetics and yields. Thus, protein aggregates produced by differentstresses exhibited different refolding behaviors. Consequently, thespecific conditions required for refolding mAb aggregates formed afterincubation at pH 3.0 may be unpredictable.

The instant invention provides methods useful in refolding monoclonalantibody aggregates produced after exposure to low pH, for example,approximately pH 3.0, as well as preparations containing such monoclonalantibody aggregates.

SUMMARY OF THE INVENTION

The present invention provides particularly effective and efficientmethods for refolding antibodies, particularly monoclonal antibodies,using high-pressure techniques (high hydrostatic pressure), as well aspreparations of refolded monoclonal antibodies refolded using suchhigh-pressure techniques. More specifically, the present invention isdirected to high pressure refolding of monoclonal antibody aggregatesproduced after exposure to low pH. The methods provide routes forovercoming the difficulties in protein therapeutic processing, byemploying the use of high pressure techniques. These methods allow forthe disaggregation, refolding, and production of high qualityantibodies, while circumventing problems that would otherwise beassociated with therapeutic protein production. The methodsadvantageously provide processing benefits associated with the use ofhigh pressure refolding of protein aggregates.

The basic method involves obtaining an antibody sample subsequent toincubation at low pH, for example, approximately pH 3.0 comprising asolution of antibody, exposing the antibody sample to high hydrostaticpressure for a period of time, and then reducing the hydrostaticpressure to atmospheric pressure, resulting in an antibody sample with ahigher content of monomeric or properly refolded antibody than prior tothe pressure exposure.

In one embodiment, the invention embraces a method for refolding asample of an antibody, where the antibody sample comprises a solution ofantibody exposed to low pH (such as pH 3). Such an antibody sample maystill be at a solution condition of low pH, in which case, the pH of thesolution is adjusted to above pH 5.0. The antibody sample is thenexposed to high hydrostatic pressure for a period of time; subsequently,the pressure is reduced to atmospheric pressure. The antibody sample,after such pressure exposure, has a higher content of monomericantibody, a higher content of properly folded antibody, or a lowercontent of aggregated antibody than the antibody sample prior to thepressure exposure. In one embodiment, the antibody sample after pressureexposure has a higher content of monomeric antibody than prior topressure exposure. In one embodiment, the antibody sample after pressureexposure has a higher content of properly refolded antibody than priorto pressure exposure. In one embodiment, the antibody sample afterpressure exposure has a lower content of aggregated antibody than priorto pressure exposure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts high pressure refolding of pH 3 induced aggregates ofCTLA-4Ig (0.5 mg/ml) as a function of pH.

FIG. 2 depicts the effect of excipients on the refolding of CTLA-4Ig lowpH induced aggregates.

FIG. 3 depicts the effect of pressure on the refolding of CTLA-4Ig lowpH induced aggregates.

FIG. 4 depicts high pressure refolding of pH 3 induced aggregates ofAlliance mAb as a function of pressure.

FIG. 5 depicts the effect of excipients on the pressure refolding of theAlliance mAb acid-induced aggregates.

FIG. 6 depicts the effect of temperature on refolding of the AlliancemAb aggregated by exposure to pH 3.0.

DETAILED DESCRIPTION OF THE INVENTION

All publications and patents mentions herein are hereby incorporated byreference in their respective entireties. The publications and patentsdisclosed herein are provided solely for their disclosure. Nothingherein is to be construed as an admission that the inventors are notentitled to antedate any publication and/or patent, including anypublication and/or patent cited herein. U.S. Pat. Nos. 6,489,450 and7,064,192, United States Patent Application Publication Nos.2004/0038333 and 2006/0188970, and International Patent ApplicationPublication No. WO 2007/062174 are specifically incorporated herein byreference in their entirety. In particular, the experimental techniquesfor refolding found in those documents are incorporated by referenceherein.

The embodiments of the present invention described below are notintended to be exhaustive or to limit the invention to the precise formsdisclosed in the following detailed description. Rather, the embodimentsare chosen and described so that others skilled in the art canappreciated and understand the principles and practices of the presentinvention.

The methods of the present invention can be used to refold monoclonalantibodies, and are especially useful for the refolding of antibodyaggregates produced after exposure to low pH. Unless otherwise stated,the following terms used in the specification and claims have themeaning(s) provided herein.

By “low pH” is meant solution conditions of about pH 1.0 to 5.0,preferably about pH 2.0 to about 4.0, more preferably about 2.5 to about3.5, more preferably about 2.8 to about 3.2, more preferably about 3.0.

As used herein, a “protein aggregate” is defined as being composed of amultiplicity of protein molecules wherein non-native noncovalentinteractions and/or non-native covalent bonds (such as non-nativeintermolecular disulfide bonds) hold the protein molecules together.Typically, but not always, an aggregate contains sufficient molecules sothat it is insoluble; such aggregates are insoluble aggregates. Thereare also oligomeric proteins which occur in aggregates in solution; suchaggregates are soluble aggregates. In addition, there is typically (butnot always) a display of at least one epitope or region on the aggregatesurface which is not displayed on the surface of native, non-aggregatedprotein. “Inclusion bodies” are a type of aggregate of particularinterest to which the present invention is applicable. Other proteinaggregates include, but are not limited to, soluble and insolubleprecipitates, soluble non-native oligomers, gels, fibrils, films,filaments, protofibrils, amyloid deposits, plaques, and dispersednon-native intracellular oligomers.

“Atmospheric,” “ambient,” or “standard” pressure is defined asapproximately 15 pounds per square inch (psi) or approximately 1 bar orapproximately 100,000 Pascals.

“Biological activity” of a protein or polypeptide as used herein, meansthat the protein or polypeptide retains at least about 10% of maximalknown specific activity as measured in an assay that is generallyaccepted in the art to be correlated with the known or intended utilityof the protein. For proteins or polypeptides intended for therapeuticuse, the assay of choice is one accepted by a regulatory agency to whichdata on safety and efficacy of the protein or polypeptide must besubmitted. In some embodiments, a protein or polypeptide having at leastabout 10% of maximal known specific activity or of the non-denaturedmolecule is “biologically active” for the purposes of the invention. Insome embodiments, the biological activity is at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 75%, or at least about 90% ofmaximal known specific activity or of the non-denatured molecule.

“Denatured,” as applied to a protein in the present context, means thatnative secondary, tertiary, and/or quaternary structure is disrupted toan extent that the protein does not have biological activity.

The “native conformation” of a protein refers to the secondary, tertiaryand/or quaternary structures of a protein in its biologically activestate.

“Refolding” in the present context means the process by which a fully orpartially denatured polypeptide adopts secondary, tertiary andquaternary structure like that of the cognate native molecule. Aproperly refolded polypeptide has biological activity that is at leastabout 10% of the non-denatured molecule, preferably biological activitythat is substantially that of the non-denatured molecule. In someembodiments, the biological activity is at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, or at least about 90% of thenon-denatured molecule. Where the native polypeptide has disulfidebonds, oxidation to form native disulfide bonds is a desired componentof the refolding process.

Antibodies which can be refolded with the methods of the inventioninclude a wide variety of polyclonal or monoclonal preparations;monoclonal antibodies are the preferred antibody embodiment forrefolding with the methods of the invention. The methods of the presentinvention are applicable to an antibody prepared via a typical processthat may include exposure to low pH, e.g., pH 3.0, during preparation.Examples of antibodies which can be refolded with the methods of theinvention, along with their indications, include, but are not limitedto: Avastin® (bevacizumab, Genentech, Inc. South San Francisco, Calif.)for treatment of metastatic colorectal cancer and non-small cell lungcancer; Bexxar® (tositumomab, Smithkline Beecham Corp., PhiladelphiaPa.) for treatment of non-Hodgkin's lymphoma; Campath® (alemtuzumab,Genzyme Corporation, Cambridge Mass.) for treatment of B-cell chroniclymphocytic leukemia; Erbitux® (cetuximab, ImClone Systems Inc., NewYork, N.Y.) for treatment of colorectal cancer; Herceptin® (trastuzumab,Genentech, Inc. South San Francisco, Calif.) for treatment of breastcancer; Humira® (adalimumab, Abbott Biotechnology Ltd., HamiltonBermuda) for treatment of rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, and Crohn's disease; Lucentis® (ranibizumab,Genentech, Inc. South San Francisco, Calif.) for treatment of wetage-related macular degeneration; Mylotarg® (gemtuzumab ozogamicin(antibody conjugated to antibiotic calicheamicin), Wyeth Corp., Madison,N.J.) for treatment of acute leukemia; Orencia® (abatacept;Bristol-Myers Squibb Co., New York, N.Y.) for treatment of rheumatoidarthritis; Orthoclone OKT3® (muromonab-CD3, Johnson & Johnson Corp., NewBrunswick, N.J.) for treatment of transplant rejection; Raptiva®(efalizumab, Genentech, Inc. South San Francisco, Calif.) for treatmentof plaque psoriasis; Remicade® (infliximab, Centocor, Inc., Malvern,Pa.) for treatment of rheumatoid arthritis, Crohn's disease, ankylosingspondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis;ReoPro® (abciximab, Eli Lilly and Co., Indianapolis Ind.) as an adjunctto percutaneous coronary intervention; Rituxan® (rituximab, Biogen IDECInc., Cambridge Mass. and Genentech, Inc. South San Francisco, Calif.)for treatment of non-Hodgkin's lymphoma and rheumatoid arthritis;Simulect® (basiliximab, Novartis AG, Basel, Switzerland) for treatmentof acute organ rejection; Soliris® (eculizumab, Alexion Pharmaceuticals,Inc., Cheshire, Conn.) for treatment of paroxysmal nocturnalhemoglobinuria; Synagis® (palivizumab, MedImmune, Inc., Gaithersburg,Md.) for treatment of respiratory syncytial virus; Tysabri®(natalizumab, Elan Pharmaceuticals, Inc., South San Francisco, Calif.)for treatment of multiple sclerosis; Vectibix® (panitumumab, ImmunexCorp., Thousand Oaks, Calif.) for treatment of metastatic colorectalcancer; Xolair® (omalizumab, Novartis AG, Basel, Switzerland) fortreatment of allergic asthma; Zenapax® (daclizumab, Roche Inc., Nutley,N.J.) for treatment of acute organ rejection; and Zevalin® (ibritumomabtiuxetan, Biogen IDEC Inc., Cambridge Mass.) for treatment of B-cellnon-Hodgkin's lymphoma.

A wide variety of techniques are known in the art for protein separationand purification, such as affinity chromatography, high-pressure liquidchromatography (HPLC), dialysis, ion exchange chromatography, sizeexclusion chromatography, reverse-phase chromatography, ammonium sulfateprecipitation, or electrophoresis. Several conditions for HPLC can bevaried for enhancing separation, such as the stationary and mobilephases. HPLC can be used with ion-exchange columns, reverse-phasecolumns, affinity columns, size-exclusion columns, and other types ofcolumns. FPLC, or “Fast Performance Liquid Chromatography,” can also beused. Gel-filtration chromatography can be used at low solventpressures. Removal of small molecules (such as chaotropes, kosmotropes,surfactants, detergents, reducing agents, oxidizing agents, or smallmolecule binding partners) from protein solutions can be achieved viadiafiltration, ultrafiltration, or dialysis.

There are several assay methods for analyzing the monomeric/refoldedcontent of antibody in a sample. A specific binding assay can beemployed, to determine the degree of binding of the antibody to aspecific binding partner. A binding assay measures the amount of activeprotein and is thus quite useful for determining the amount offunctional protein.

Several methods based on physical parameters are available for analyzingand quantitating aggregated proteins, such as antibodies, anddetermining amounts of aggregated proteins and monomeric proteins. Anexcellent overview of several methods of analysis of macromolecules isfound in Cantor, C. R. and P. R. Schimmel, Biophysical Chemistry PartII: Techniques for the Study of Biological Structure and Function, W.H.Freeman & Co., New York: 1980. Other general techniques are described inUS Patent Application Publication No. 2003/0022243.

The use of analytical ultracentrifugation for characterization ofaggregation of protein therapeutics is specifically discussed in Philo,J. S., American Biotechnology Laboratory, page 22, October 2003.Experiments that can be performed using analytical ultracentrifugationinclude sedimentation velocity and sedimentation equilibriumexperiments, which can be performed to determine whether multiplesolutes exist in a solution (e.g., monomer, dimer, trimer, etc.) andprovide an estimate of molecular weights for the solutes.

Size-exclusion chromatography and gel permeation chromatography can beused to estimate molecular weights and aggregation numbers of proteins,as well as for separation of different aggregates. See references suchas Wu, C.-S. (editor), Handbook of Size Exclusion Chromatography andRelated Techniques, Second Edition (Chromatographic Science), MarcelDekker: New York, 2004 (particularly chapter 15 at pages 439-462 byBaker et al., “Size Exclusion Chromatography of Proteins”) and Wu, C.-S.(editor), Column Handbook for Size Exclusion Chromatography, San Diego:Academic Press, 1999 (particularly Chapters 2 and 18).

Field flow fractionation, which relies on a field perpendicular to aliquid stream of molecules, can also be used to analyze and separateaggregated proteins such as protein monomers, dimers, trimers, etc. SeeZhu et al., Anal. Chem. 77:4581 (2005); Litzen et al., Anal. Biochem.212:469 (1993); and Reschiglian et al., Trends Biotechnol. 23:475(2005).

Light scattering methods, such as methods using laser light scattering(often in conjunction with size-exclusion chromatography or othermethods) can also be used to estimate the molecular weight of proteins,including protein aggregates; see, for example, Mogridge, J., MethodsMol Biol. 261:113 (2004) and Ye, H., Analytical Biochem. 356:76 (2006).Dynamic light scattering techniques are discussed in Pecora, R., ed.,Dynamic Light Scattering: Applications of Photon CorrelationSpectroscopy, New York: Springer Verlag, 2003 and Berne, B. J. andPecora, R., Dynamic Light Scattering: With Applications to Chemistry,Biology, and Physics, Mineola, N.Y.: Dover Publications, 2000. Laserlight scattering is discussed in Johnson, C. S. and Gabriel, D. A.,Laser Light Scattering, Mineola, N.Y.: Dover Publications, 1995, andother light scattering techniques which can be applied to determineprotein aggregation are discussed in Kratochvil, P., Classical LightScattering from Polymer Solutions, Amsterdam: Elsevier, 1987.

Light obscuration can also be used to measure protein aggregation; seeSeefeldt et al., Protein Sci. 14:2258 (2005); Kim et al., J. Biol. Chem.276: 1626 (2001); and Kim et al., J. Biol. Chem. 277: 27240 (2002).

Fluorescence spectroscopy, such as fluorescence anisotropy spectroscopy,can be used to determine the presence of protein aggregates.Fluorescence probes (dyes) can be covalently or non-covalently bound tothe aggregate to aid in analysis of aggregates (see, e.g., Lindgren etal., Biophys. J. 88: 4200 (2005)), US Patent Application Publication2003/0203403), or Royer, C. A., Methods Mol. Biol. 40:65 (1995).Internal tryptophan residues can also be used to detect proteinaggregation; see, e.g., Dusa et al., Biochemistry 45:2752 (2006).

Many methods of gel electrophoresis can be employed to analyze proteinsand protein aggregation. One of the most common methods of gelelectrophoresis is polyacrylamide gel electrophoresis (PAGE). If anaggregate is covalently linked, denaturing PAGE (using, e.g., sodiumdodecyl sulfate) can be employed. Native PAGE (non-denaturing PAGE) canbe used to study non-covalently linked aggregates. See, e.g., Hermelinget al. J. Phar. Sci. 95:1084-1096 (2006); Kilic et al., Protein Sci.12:1663 (2003); Westermeier, R., Electrophoresis in Practice: A Guide toMethods and Applications of DNA and Protein Separations 4^(th) edition,New York: John Wiley & Sons, 2005; and Hames, B. D. (Ed.), GelElectrophoresis of Proteins: A Practical Approach, 3^(rd) edition, NewYork: Oxford University Press, USA, 1998.

Gas-phase electrophoretic mobility molecular analysis (GEMMA) (seeBacher et al., J. Mass Spectrom. 36:1038 (2001), Kaufman et al., Anal.Chem. 68:1895 (1996) and Kaufman et al., Anal. Biochem. 259:195 (1998)),a combination of electrophoresis in the gas phase and mass spectrometry,provides another method of analyzing protein complexes and aggregates.

Nuclear magnetic resonance spectroscopic techniques can be used toestimate hydrodynamic parameters related to protein aggregation. See,for example, James, T. L. (ed.), Nuclear Magnetic Resonance ofBiological Macromolecules, Part C, Volume 394: Methods in Enzymology,San Diego: Academic Press, 2005; James, T. L., Dotsch, V. and Schmitz,U. (eds.), Nuclear Magnetic Resonance of Biological Macromolecules, PartA (Methods in Enzymology, Volume 338) and Nuclear Magnetic Resonance ofBiological Macromolecules, Part B (Methods in Enzymology, Volume 339),San Diego: Academic Press, 2001, and Mansfield, S. L. et al., J. Phys.Chem. B, 103:2262 (1999). Linewidths, correlation times, and relaxationtimes are among the parameters that can be measured to estimate tumblingtime in solution, which can then be correlated with the state of proteinaggregation. Electron paramagnetic resonance (EPR or ESR) can also beused to determine aggregation states; see, e.g., Squier et al., J. Biol.Chem. 263:9162 (1988).

Reverse-phase high-pressure liquid chromatography (RP-HPLC) can also beused to determine monomer/aggregate content of protein preparations,although this method must be used cautiously, as the conditions used forRP-HPLC may alter the amount of aggregate present in the originalsample.

In one embodiment of the invention, analytical ultracentrifugation isused for the comparison of aggregates in pressure-treated and untreatedsamples. In another embodiment of the invention, size exclusionchromatography is used for the comparison of aggregates inpressure-treated and untreated samples. In another embodiment of theinvention, field flow fractionation is used for the comparison ofaggregates in pressure-treated and untreated samples. In anotherembodiment of the invention, light scattering analysis is used for thecomparison of aggregates in pressure-treated and untreated samples. Inanother embodiment of the invention, light obscuration analysis is usedfor the comparison of aggregates in pressure-treated and untreatedsamples. In another embodiment of the invention, fluorescencespectroscopy is used for the comparison of aggregates inpressure-treated and untreated samples. In another embodiment of theinvention, gel electrophoresis is used for the comparison of aggregatesin pressure-treated and untreated samples. In another embodiment of theinvention, GEMMA is used for the comparison of aggregates inpressure-treated and untreated samples. In another embodiment of theinvention, nuclear magnetic resonance spectroscopy is used for thecomparison of aggregates in pressure-treated and untreated samples. Inanother embodiment of the invention, electron paramagnetic resonancespectroscopy is used for the comparison of aggregates inpressure-treated and untreated samples. In another embodiment of theinvention, reverse-phase chromatography is used for the comparison ofaggregates in pressure-treated and untreated samples.

Several conditions can be adjusted for optimal protein refolding:protein concentration; agitation; temperature; reduction of pressure andcombinations thereof.

The concentration of protein can be adjusted for optimal proteinrefolding. One advantage of high-pressure protein refolding is that muchhigher concentrations of protein can be used as compared to chemicalrefolding techniques. Protein concentrations of at least about 0.1mg/ml, at least about 1.0 mg/ml, at least about 5.0 mg/ml, at leastabout 10 mg/ml, or at least about 20 mg/ml can be used. Protein in thesample may be present in a concentration of from about 0.001 mg/ml toabout 300 mg/ml. Thus, in some embodiments the protein is present in aconcentration of from about 0.001 mg/ml to about 250 mg/ml, from about0.001 mg/ml to about 200 mg/ml, from about 0.001 mg/ml to about 150mg/ml, from about 0.001 mg/ml to about 100 mg/ml, from about 0.001 mg/mlto about 50 mg/ml, from about 0.001 mg/ml to about 30 mg/ml, from about0.05 mg/ml to about 300 mg/ml, from about 0.05 mg/ml to about 250 mg/ml,from about 0.05 mg/ml to about 200 mg/ml, from about 0.05 mg/ml to about150 mg/ml, from about 0.05 mg/ml to about 100 mg/ml, from about 0.05mg/ml to about 50 mg/ml, from about 0.05 mg/ml to about 30 mg/ml, fromabout 10 mg/ml to about 300 mg/ml, from about 10 mg/ml to about 250mg/ml, from about 10 mg/ml to about 200 mg/ml, from about 10 mg/ml toabout 150 mg/ml, from about 10 mg/ml to about 100 mg/ml, from about 10mg/ml to about 50 mg/ml, from about 10 mg/ml to about 30 mg/ml, fromabout 0.1 mg/ml to about 100 mg/ml, from about 0.1 mg/ml to about 10mg/ml, from about 1 mg/ml to about 100 mg/ml, from about 1 mg/ml toabout 10 mg/ml, from about 10 mg/ml to about 100 mg/ml, or from about 50mg/ml to about 100 mg/ml can be used.

As used in the present context the phrase “a period of time sufficientto form biologically active protein” and cognates thereof refer to thetime needed for the protein aggregates to be disaggregated and to adopta conformation where the protein is biologically active. Typically, thetime sufficient for solubilization is about 15 minutes to about 50hours, or possibly longer depending on the particular protein, (e.g., aslong as necessary for the protein; for example, up to about 1 week,about 5 days, about 4 days, about 3 days, etc.). Thus, in someembodiments of the methods, the time sufficient for formation ofbiologically active protein may be from about 2 to about 30 hours, fromabout 2 to about 24 hours, from about 2 to about 18 hours, from about 1to about 10 hours, from about 1 to about 8 hours, from about 1 to about6 hours, from about 2 to about 10 hours, from about 2 to about 8 hours,from about 2 to about 6 hours, or about 2 hours, about 6 hours, about 10hours, about 16 hours, about 20 hours, or about 30 hours, from about 2to about 10 hours, from about 2 to about 8 hours, from about 2 to about6 hours, from about 12 to about 18 hours, or from about 10 to about 20hours.

The sample comprising protein aggregates or denatured protein istypically an aqueous solution or aqueous suspension. The sample may alsoinclude other components. These additional components may be one or moreadditional agents including: one or more stabilizing agents, one or morebuffering agents, one or more surfactants, one or more salts, one ormore chaotropes, or combinations of two or more of the foregoing.

The amounts of the additional agents will vary depending on theselection of the protein, however, the effect of the presence (andamount) or absence of each additional agent or combinations of agentscan be determined and optimized using the teachings provided herein.

Exemplary additional agents include, but are not limited to, buffers(examples include, but are not limited to, phosphate buffer, boratebuffer, carbonate buffer, citrate buffer, HEPES, MEPS), salts (examplesinclude, but are not limited to, the chloride, sulfate, and carbonatesalts of sodium, zinc, calcium, ammonium and potassium), chaotropes(examples include, but are not limited to, urea, guanidinehydrochloride, guanidine sulfate and sarcosine), and stabilizing agents(e.g., preferential excluding compounds, etc.).

Non-specific protein stabilizing agents act to favor the most compactconformation of a protein. Such agents include, but are not limited to,one or more free amino acids, one or more preferentially excludingcompounds, kosmotropes, trimethylamine oxide, cyclodextrans, molecularchaperones, and combinations of two or more of the foregoing.

Amino acids can be used to prevent reaggregation and facilitate thedissociation of hydrogen bonds. Typical amino acids that can be used,but not limited to, are arginine, lysine, proline, glycine, histidine,and glutamine or combinations of two or more of the foregoing. In someembodiments, the free amino acid(s) is present in a concentration ofabout 0.1 mM to about the solubility limited of the amino acid, and insome variations from about 0.1 mM to about 2 M. The optimalconcentration is a function of the desired protein and should favor thenative conformation.

Preferentially excluding compounds can be used to stabilize the nativeconfirmation of the protein of interest. Possible preferentiallyexcluding compounds include, but are not limited to, sucrose, hexyleneglycol, sugars (e.g., sucrose, trehalose, dextrose, mannose), andglycerol. The range of concentrations that can be use are from 0.1 mM tothe maximum concentration at the solubility limit of the specificcompound. The optimum preferential excluding concentration is a functionof the protein of interest.

In particular embodiments, the preferentially excluding compound is oneor more sugars (e.g., sucrose, trehalose, dextrose, mannose orcombinations of two or more of the foregoing). In some embodiments, thesugar(s) is present in a concentration of about 0.1 mM to about thesolubility limit of the particular compound. In some embodiments, theconcentration is from about 0.1 mM to about 2M, from about 0.1 mM toabout 1.5M, from about 0.1 mM to about 1M, from about 0.1 mM to about0.5M, from about 0.1 mM to about 0.3M, from about 0.1 mM to about 0.2 M,from about 0.1 mM to about 0.1 mM, from about 0.1 mM to about 50 mM,from about 0.1 mM to about 25 mM, or from about 0.1 mM to about 10 mM.When present as a percentage of solution (w/w and/or w/v), theconcentration can be about 0.1% to about 20%, about 1% to about 20%,about 1% to about 15%, about 5% to about 20%, about 5% to about 15%,about 8% to about 12%, or about 10%.

In some embodiments, the stabilizing agent is one or more of sucrose,trehalose, glycerol, betaine, amino acid(s), or trimethylamine oxide.

In certain embodiments, the stabilizing agent is a cyclodextran. In someembodiments, the cyclodextran is present in a concentration of about 0.1mM to about the solubility limit of the cyclodextran. In somevariations, the cyclodextran is present in a concentration from about0.1 mM to about 2 M.

In certain embodiments, the stabilizing agent is a molecular chaperone.In some embodiments, the molecular chaperone is present in aconcentration of about 0.01 mg/ml to 10 mg/ml.

A single stabilizing agent maybe be used or a combination of two or morestabilizing agents (e.g., at least two, at least three, or 2 or 3 or 4stabilizing agents). Where more than one stabilizing agent is used, thestabilizing agents may be of different types, for example, at least onepreferentially excluding compound and at least one free amino acid, atleast one preferentially excluding compound and betaine, etc.

Buffering agents may be present to maintain a desired pH value or pHrange. Numerous suitable buffering agents are known to the skilledartisan and should be selected based on the pH that favors (or whichdoes not disfavor) the native conformation of the protein of interest.Either inorganic or organic buffering agents may be used. Suitableconcentrations are known to the skilled artisan and should be optimizedfor the methods as described herein according to the teaching providedbased on the characteristics of the desired protein.

Thus, in some embodiments, at least one inorganic buffering agent isused (e.g., phosphate, carbonate, etc.). In certain embodiments, atleast one organic buffering agent is used (e.g., citrate, acetate, Tris,MOPS, MES, HEPES, etc.) Additional organic and inorganic bufferingagents are well known to the art.

In some embodiments, the one or more buffering agents is phosphatebuffer, borate buffer, carbonate buffer, citrate buffer, HEPES, MEPS,MOPS, MES, or acetate buffer.

In some embodiments, the one or more buffering agents is phosphatebuffers, carbonate buffers, citrate, Tris, MOPS, MES, acetate or HEPES.

A single buffering agent maybe be used or a combination of two or morebuffering agents (e.g., at least two, at least 3, or 2 or 3 or 4buffering agents).

A “surfactant” as used in the present context is a surface activecompound which reduces the surface tension of water.

Surfactants are used to improve the solubility of certain proteins.Surfactants should generally be used at concentrations above or belowtheir critical micelle concentration (CMC), for example, from about 5%to about 20% above or below the CMC. However, these values will varydependent upon the surfactant chosen, for example, surfactants such as,beta-octylgluco-pyranoside may be effective at lower concentrationsthan, for example, surfactants such as TWEEN-20 (polysorbate 20). Theoptimal concentration is a function of each surfactant, which has itsown CMC.

Useful surfactants include nonionic (including, but not limited to,t-octylphenoxypolyethoxy-ethanol and polyoxyethylene sorbitan), anionic(e.g., sodium dodecyl sulfate) and cationic (e.g., cetylpyridiniumchloride) and amphoteric agents. Suitable surfactants include, but arenot limited to deoxycholate, sodium octyl sulfate, sodium tetradecylsulfate, polyoxyethylene ethers, sodium cholate,octylthioglucopyranoside, n-octylglucopyranoside, alkyltrimethylammoniumbromides, alkyltrimethyl ammonium chlorides, and sodium bis(2ethylhexyl) sulfosuccinate. In some embodiments the surfactant may bepolysorbate 80, polysorbate 20, sarcosyl, Triton X-100,β-octyl-gluco-pyranoside, or Brij 35.

In some embodiments the one or more surfactant may be a polysorbate,polyoxyethylene ether, alkyltrimethylammonium bromide, pyranosides orcombination of two or more of the foregoing. In certain embodiments, theone or more surfactant may be α-octyl-gluco-pyranoside, Brij 35, or apolysorbate.

In certain embodiments the one or more surfactant may be octyl phenolethoxylate, β-octyl-gluco-pyranoside, polyoxyethyleneglycol dodecylether, sarcosyl, sodium dodecyl sulfate, polyethoxysorbitan,deoxycholate, sodium octyl sulfate, sodium tetradecyl sulfate, sodiumcholate, octylthioglucopyranoside, n-octylglucopyranoside, sodiumbis(2-ethylhexyl) sulfosuccinate or combinations of two or more of theforegoing. A single surfactant maybe be used or a combination of two ormore surfactants (e.g., at least two, at least 3, or 2 or 3 or 4surfactants).

Chaotropic agents (also referred to as a “chaotrope”) are compounds,including, without limitation, guanidine, guanidine hydrochloride(guanidinium hydrochloride, GdmHCl), guanidine sulfate, urea, sodiumthiocyanate, and/or other compounds which disrupt the noncovalentintermolecular bonding within the protein, permitting the polypeptidechain to assume a substantially random conformation

Chaotropic agents may be used in concentration of from about 10 mM toabout 8 M. The optimal concentration of the chaotropic agent will dependon the desired protein as well as on the particular chaotropes selected.The choice of particular chaotropic agent and determination of optimalconcentration can be optimized by the skilled artisan in view of theteachings provided herein.

In some embodiments, the concentration of the chaotropic agent will be,for example, from about 10 mM to about 8 M, from about 10 mM to about 7M, from about 10 mM to about 6 M, from about 0.1 M to about 8 M, fromabout 0.1 M to about 7 M, from about 0.1 M to about 6 M, from about 0.1M to about 5 M, from about 0.1 M to about 4 M, from about 0.1 M to about3 M, from about 0.1 M to about 2 M, from about 0.1 M to about 1 M, fromabout 10 mM to about 4 M, from about 10 mM to about 3 M, from about 10mM to about 2 M, from about 10 mM to about 1 M, or about, 10 mM, about50 mM, about 75 mM, about 0.1 M, about 0.5 M, about 0.8 M, about 1 M,about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, orabout 8 M.

When used in the present methods, it is often advantageous to usechaotropic agents in non-denaturing concentrations to facilitate thedissociation of non-covalent interactions. While a non-denaturingconcentration will vary depending on the desired protein, the range ofnon-denaturing concentrations is typically from about 0.1 to about 4 M.In some embodiments the concentration is from about 0.1 M to about 2 M.

In certain embodiments, guanidine hydrochloride or urea are thechaotropic agents.

A single chaotropic agent maybe be used or a combination of two or morechaotropic agents (e.g., at least two, at least 3, or 2 or 3 or 4chaotropic agents).

Agitation is another aspect that may be manipulated. Protein solutionscan be agitated before and/or during refolding. Agitation can beperformed by methods including, but not limited to, ultrasound energy(sonication), mechanical stirring, mechanical shaking, pumping throughmixers, or via cascading solutions. Agitation may be performed for anylength of time, such as the entire period of high-pressure treatment, orfor one or more periods of about 1 to about 60 minutes duringhigh-pressure treatment, such as about 1 minute, about 5 minutes, about10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, orabout 60 minutes.

Yet another aspect that may be manipulated is temperature. The methodsdescribed herein can be performed at a range of temperature values,depending on the particular protein of interest. The optimaltemperature, in concert with other factors, can be optimized asdescribed herein. Proteins can be refolded at various temperatures,including at about room temperature, about 20° C., about 25° C., about30° C., about 37° C., about 50° C., about 75° C., about 100° C., about125° C., or ranges of from about 20 to about 125° C., about 25 to about125° C., about 25 to about 100° C., about 25 to about 75° C., about 25to about 50° C., about 50 to about 125° C., about 50 to about 100° C.,about 50 to about 75° C., about 75 to about 125° C., about 5 to about100° C., or about 100 to about 125° C.

In some embodiments of the methods, the temperature can range from about0° C. to about 100° C. without adversely affecting the protein ofinterest. Thus in certain embodiments, the temperature may be from about0° C. to about 75° C., from about 0° C. to about 55° C., from about 0°C. to about 35° C., from about 0° C. to about 25° C., from about 20° C.to about 75° C., from about 20° C. to about 65° C., from about 20° C. toabout 35° C., or from about 20° C. to about 25° C.

Although increased temperatures are often used to cause aggregation ofproteins, when coupled with increased hydrostatic pressure it has beenfound that increased temperatures can enhance refolding recoverieseffected by high pressure treatment, provided that the temperatures arenot so high as to cause irreversible denaturation. Generally, theincreased temperature for refolding should be about 20° C. lower thanthe temperatures at which irreversible loss of activity occurs.Relatively high temperatures (for example, about 60° C. to about 125°C., about 80° C. to about 110° C., including about 100° C., about 105°C., about 110° C., about 115° C., about 120° C. and about 125° C.) maybe used while the solution is under pressure, as long as the temperatureis reduced to a suitably low temperature before depressurizing. Such asuitably low temperature is defined as one below which thermally-induceddenaturation or aggregation occurs at atmospheric conditions.

“High pressure” or “high hydrostatic pressure,” for the purposes of theinvention is defined as pressures of from about 500 bar to about 10,000bar.

In some embodiments, the increased hydrostatic pressure may be fromabout 500 bar to about 5000 bar, from about 500 bar to about 4000 bar,from about 500 bar to about 2000 bar, from about 500 bar to about 2500bar, from about 500 bar to about 3000 bar, from about 500 bar to about6000 bar, from about 1000 bar to about 5000 bar, from about 1000 bar toabout 4000 bar, from about 1000 bar to about 2000 bar, from about 1000bar to about 2500 bar, from about 1000 bar to about 3000 bar, from about1000 bar to about 6000 bar, from about 1500 bar to about 5000 bar, fromabout 1500 bar to about 3000 bar, from about 1500 bar to about 4000 bar,from about 1500 bar to about 2000 bar, from about 2000 bar to about 5000bar, from about 2000 bar to about 4000 bar, from about 2000 bar to about3000 bar, or about 1000 bar, about 1500 bar, about 2000 bar, about 2500bar, about 3000 bar, about 3500 bar, about 4000 bar, about 5000 bar,about 6000 bar, about 7000 bar, about 8000 bar, or about 9000 bar.

Reduction of pressure is another parameter that can be manipulated.Where the reduction in pressure is performed in a continuous manner, therate of pressure reduction can be constant or can be increased ordecreased during the period in which the pressure is reduced. In somevariations, the rate of pressure reduction is from about 5000 bar/1 secto about 5000 bar/4 days (or about 3 days, about 2 days, or about 1day). Thus in some variations the rate of pressure reduction can beperformed at a rate of from about 5000 bar/1 sec to about 5000 bar/80hours, from about 5000 bar/1 sec to about 5000 bar/72 hours, from about5000 bar/1 sec to about 5000 bar/60 hours, from about 5000 bar/1 sec toabout 5000 bar/50 hours, from about 5000 bar/1 sec to about 5000 bar/48hours, from about 5000 bar/1 sec to about 5000 bar/32 hours, from about5000 bar/1 sec to about 5000 bar/24 hours, from about 5000 bar/1 sec toabout 5000 bar/20 hours, from about 5000 bar/1 sec to about 5000 bar/18hours, from about 5000 bar/1 sec to about 5000 bar/16 hours, from about5000 bar/1 sec to about 5000 bar/12 hours, from about 5000 bar/1 sec toabout 5000 bar/8 hours, from about 5000 bar/1 sec to about 5000 bar/4hours, from about 5000 bar/1 sec to about 5000 bar/2 hours, from about5000 bar/1 sec to about 5000 bar/1 hour, from about 5000 bar/1 sec toabout 1000 bar/min, about 5000 bar/1 sec to about 500 bar/min, about5000 bar/1 sec to about 300 bar/min, about 5000 bar/1 sec to about 250bar/min, about 5000 bar/1 sec to about 200 bar/min, about 5000 bar/1 secto about 150 bar/min, about 5000 bar/1 sec to about 100, about 5000bar/1 sec to about 80 bar/min, about 5000 bar/1 sec to about 50 bar/min,or about 5000 bar/1 sec to about 10 bar/min. For example, about 10bar/min, about 250 bar/5 minute, about 500 bar/5 minutes, about 1000bar/5 minutes, about 250 bar/5 minutes, 2000 bar/50 hours, 3000 bar/50hours, 40000 bar/50 hours, etc. In some embodiments, the pressurereduction may be approximately instantaneous, as in where pressure isreleased by simply opening the device in which the sample is containedand immediately releasing the pressure.

Where the reduction in pressure is performed in a stepwise manner, theprocess comprises dropping the pressure from the highest pressure usedto at least a secondary level that is intermediate between the highestlevel and atmospheric pressure. The goal is to provide a hold period ator about this intermediate pressure zone that permits a protein to adopta desired conformation.

In some embodiments, where there are at least two stepwise pressurereductions there may be a hold period at a constant pressure betweenintervening steps. The hold period may be from about 10 minutes to about50 hours (or longer, depending on the nature of the protein ofinterest). In some embodiments, the hold period may be from about 2 toabout 30 hours, from about 2 to about 24 hours, from about 2 to about 18hours, from about 1 to about 10 hours, from about 1 to about 8 hours,from about 1 to about 6 hours, from about 2 to about 10 hours, fromabout 2 to about 8 hours, from about 2 to about 6 hours, or about 2hours, about 6 hours, about 10 hours, about 20 hours, or about 30 hours,from about 2 to about 10 hours, from about 2 to about 8 hours, or fromabout 2 to about 6 hours.

In some variations, the pressure reduction includes at least 2 stepwisereductions of pressure (e.g., highest pressure reduced to a secondpressure reduced atmospheric pressure would be two stepwise reductions).In other embodiments the pressure reduction includes more than 2stepwise pressure reductions (e.g., 3, 4, 5, 6, etc.). In someembodiments, there is at least 1 hold period. In certain embodimentsthere is more than one hold period (e.g., at least 2, at least 3, atleast 4, at least 5 hold periods).

In some variations of the methods the constant pressure after an initialstepwise reduction may be at a hydrostatic pressure of from about 500bar to about 5000 bar, from about 500 bar to about 4000 bar, from about500 bar to about 2000 bar, from about 1000 bar to about 4000 bar, fromabout 1000 bar to about 3000 bar, from about 1000 bar to about 2000 bar,from about 1500 bar to about 4000 bar, from about 1500 bar to about 3000bar, from about 2000 bar to about 4000 bar, or from about 2000 bar toabout 3000 bar.

In particular variations, constant pressure after the stepwise reductionis from about four-fifths of the pressure immediately prior to thestepwise pressure reduction to about one-tenth of prior to the stepwisepressure reduction. For example, constant pressure is at a pressure offrom about four-fifths to about one-fifth, from about two-thirds toabout one-tenth, from about two-thirds to about one-fifth, from abouttwo-thirds to about one-third, about one-half, or about one-quarter ofthe pressure immediately prior to the stepwise pressure reduction. Wherethere is more than one stepwise pressure reduction step, the pressurereferred to is the pressure immediately before the last pressurereduction (e.g., where 2000 bar is reduced to 1000 bar is reduced to 500bar, the pressure of 500 bar is one-half of the pressure immediatelypreceding the previous reduction (1000 bar)).

Where the pressure is reduced in a stepwise manner, the rate of pressurereduction (e.g., the period of pressure reduction prior to and after thehold period) may be in the same range as that rate of pressure reductiondescribed for continuous reduction (e.g., in a non-stepwise manner). Inessence, stepwise pressure reduction is the reduction of pressure in acontinuous manner to an intermediate constant pressure, followed by ahold period and then a further reduction of pressure in a continuousmanner. The periods of continuous pressure reduction prior to and aftereach hold period may be the same continuous rate for each period ofcontinuous pressure reduction or each period may have a differentreduction rate. In some variations, there are two periods of continuouspressure reduction and a hold period. In certain embodiments, eachcontinuous pressure reduction period has the same rate of pressurereduction. In other embodiments, each period has a different rate ofpressure reduction. In particular embodiments, the hold period is fromabout 8 to about 24 hours. In some embodiments, the hold period is fromabout 12 to about 18 hours. In particular embodiments, the hold periodis about 16 hours.

Various combinations and permutations of the condition above, such asagitation of the protein under high pressure at an elevated temperaturein the presence of chaotropes and redox reagents, can be employed asdesired for optimization of refolding yields.

Optimization of reaction conditions for solubilization and refolding inthe context of the methods described herein are a function of thecharacteristics of both the target antibody and any other components orparameters of the solution. In standard optimization experiments, theinfluence of pressure, pH, temperature, ionic strength, surfactants,chaotropes, stabilizing agents, and refolding time on refolding shouldbe tested. Once the key process parameters are identified, a centralcomposite design can be used to optimize the appropriate conditions foreach parameter. Guidance regarding typical ranges for the variousparameters is provided in more detail below.

Initial studies can be conducted to screen the effect of solutionconditions, solution pH, and high pressure treatment on thesolubilization and/or refolding of proteins. Screening studies aretypically conducted, but not limited to, empirical screens that examinestep-wise the effect of processing conditions on yields. Synergisticeffects between different parameters are not examined in these screeningstudies. Exemplary screening studies that can be conducted are asdescribed for the cases of recombinant placental bikunin, recombinantgrowth hormone, and malaria pfs48 (see e.g., Seefeldt et al. ProteinScience, v13 (10), 2639-2650 2004, St. John et al., Journal ofBiological Chemistry, v276 (50), 46856-46864, 2001, Seefeldt, “Highpressure refolding of protein aggregates: efficacy and thermodynamics,”Dept. of Chemical and Biological Engineering Thesis, (2004), thedisclosures of which are herein incorporated by reference in theirentirety, particularly with respect to the screening studies describedtherein). High pressure refolding studies of bikunin and growth hormonedemonstrate the step-wise screening process for solution conditions (pH5-9), temperature (0-60° C.), ionic strength (0-160 mM NaCl),non-denaturing concentrations of chaotropes (0-1.0 M urea or 0-2.0 Mguanidine) and refolding time (0-24 hours). Studies can be conducted atabout 2000 bar, about 2100 bar, about 2150 bar, etc. and compared tosamples treated at atmospheric pressure. Other parameters, includingthose described herein, that can be screened include, but are notlimited to, the presence and amount of stabilizing agents, surfactants,salts, etc., as described herein. It should be noted that statisticalanalysis of variance (ANOVA's) can be used to rapidly screen whichsolution parameters affect refolding yields. In addition to the teachingprovided herein, U.S. 2004/0038333, Seefeldt et al. Protein Science, v13(10), 2639-2650 2004, and St. John et al., Biotechnology Progress, v18,(3), 565-571, 2002 (incorporated herein by reference in their entirety)also provide guidance regarding empirical screening procedures fordetermining the optimal solubilization and refolding conditions.

In this manner, the skilled artisan can determine the effect ofprocessing conditions on the refolding of protein aggregates through theuse of high pressure. It has been shown in the literature that refoldingreactions can have interactions between the process conditions, whichprevents single-variable screening from effectively optimizing theprocess. For instance, pH affects protein conformation stability,protein colloidal stability, and disulfide bond formation kinetics. Toeffectively optimize the effect of pH, or any other process parameter,studies need to be conducted to account for interactions. In theseinstances, statistical experimental designs need to be employed. Asdescribed herein, solubilization is also examined as a function of urea,by step-wise analysis in a range from 0-4.5 M urea at pH 8.0. Once thesignificant parameters are identified, a face-centered statisticaldesigned experiment is used to optimize the refolding conditions, takinginto account interactions.

After initial optimization studies are performed for the protein ofinterest, more granular optimization can be used to determine theoptimal conditions for performing the solubilization and refoldingprocesses. This process can generally be described as an experimentaloptimization that takes into account synergistic interactions betweenthe critical parameters identified in the initial step-wise studies. Aneffective method for conducting these studies involves using a three orfive level central composite statistical analysis, which takes intoaccount interactions between the reaction parameters while minimizingthe required number of experiments.

Another useful aid for optimizing conditions and/or monitoringsolubilization or refolding is in situ spectroscopic measurement ofsamples under pressure, a well-known process for examining polypeptidestability under pressure. Using high pressure spectroscopic techniquesto observe aggregate dissolution under pressure will help determine theoptimal pressure ranges for recovering proteins from aggregates. Custommade high pressure cells have been routinely used for high pressureunfolding studies and can be adapted for use in high pressuredisaggregation and refolding. Additional guidance for the skilledartisan may also be found in Paladini and Weber, Biochemistry, 20 (9),2587-2593 (1981) and Seefeldt et al. Protein Science, 13 (10), 2639-2650(2004), incorporated by reference herein in their entirety.

Methods that can be employed to monitor the optimization of variousparameters include Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD) spectroscopy (far and/or near UV), UVspectroscopy, measurement of total protein concentrations (e.g., BCAassay method (Pierce Chemical Co., Rockford, Ill.), etc), activityassays to measure the activity of the target polypeptide,electrophoretic gels with molecular weight markers to visualize theappearance of native protein under various conditions, HPLC analysis ofsoluble polypeptide fractions, etc.

Suitable devices for performing high pressure spectroscopy can beobtained commercially (e.g., such as fluorescence cells available fromISS Inc., Champaign, Ill. or fluorescence/ultraviolet absorbance cellsavailable from BaroFold Inc., Boulder, Colo.) or can be fabricated bythe skilled artisan. For example, Randolph et al., U.S. PatentApplication Publication No. 2004/0038333, incorporated by referenceherein in its entirety, described a high-pressure W spectroscopy cellmade of stainless steel, sealed with Btma-N 90 durometer o-rings andwith an optical port diameter of 6 mm and pathlength of 7.65 mm. Thecell utilized cylindrical sapphire windows (16 mm diameter, 5.1 mmthick) and was capable of experiments up to 250 MPa. Separation of thesample from the pressure transmitting fluid was facilitated by a pistondevice external to the cell.

Commercially available high pressure devices and reaction vessels, suchas those described in the examples, may be used to achieve thehydrostatic pressures in accordance with the methods described herein(see BaroFold Inc., Boulder, Colo.). Additionally devices, vessels andother materials for carrying out the methods described herein, as wellas guidance regarding the performing increased pressure methods, aredescribed in detail in U.S. Pat. No. 6,489,450, which is incorporatedherein in its entirety. The skilled artisan is particularly directed tocolumn 9, lines 39-62 and Examples 2-4. International Pat. App. Pub. No.WO 02/062827, incorporated herein in its entirety, also provides theskilled artisan with detailed teachings regarding devices and usethereof for high hydrostatic pressure solubilization of aggregatesthroughout the specification. Particular devices and teachings regardingthe use of high pressure devices are also provided in InternationalPatent Application Publication No. WO 2007/062174, which is incorporatedby reference herein in its entirety.

Multiple-well sample holders may be used and can be conveniently sealedusing self-adhesive plastic covers. The containers, or the entiremultiple-well sample holder, may then be placed in a pressure vessel,such as those commercially available from the Flow International Corp.or High Pressure Equipment Co. The remainder of the interior volume ofthe high-pressure vessel may than be filled with water or other pressuretransmitting fluid.

Mechanically, there are two primary methods of high-pressure processing:batch and continuous. Batch processes simply involve filling a specifiedchamber, pressurizing the chamber for a period of time, anddepressurizing the batch. In contrast, continuous processes constantlyfeed aggregates into a pressure chamber and soluble, refolded proteinsmove out of the pressure chamber. In both set ups, good temperature andpressure control is essential, as fluctuations in these parameters cancause inconsistencies in yields. Both temperature and pressure should bemeasured inside the pressure chamber and properly controlled.

There are many methods for handling batch samples depending upon thespecific stability issues of each target protein. Samples can be loadeddirectly into a pressure chamber, in which case the aqueous solutionand/or suspension would be used as the pressure medium.

Alternately, samples can be loaded into any variety of sealed, flexiblecontainers, including those described herein. This allows for greaterflexibility in the pressure medium, as well as the surfaces to which thesample is exposed. Sample vessels could conceivably even act to protectthe desired protein from chemical degradation (e.g., oxygen scavengingplastics are available).

With continuous processing, small volumes under pressure can be used torefold large volumes of the sample. In addition, using an appropriatefilter on the outlet of a continuous process will selectively releasesoluble desired protein from the chamber while retaining both solubleand insoluble aggregates.

Pressurization is a process of increasing the pressure (usually fromatmospheric or ambient pressure) to a higher pressure. Pressurizationtakes place over a predetermined period of time, ranging from 0.1 secondto 10 hours. Such times include 1 second, 2 seconds, 5 seconds, 10seconds, 20 seconds, 1 minute, 2 minutes, 5 minutes, to minutes, 30minutes, 60 minutes, 2 hours, 3 hours, 4 hours, and 5 hours.

Depressurization is a process of decreasing the pressure, from a highpressure, to a lower pressure (usually atmospheric or ambient pressure).Depressurization takes place over a predetermined period of time,ranging from 10 seconds to 10 hours, and may be interrupted at one ormore points to permit optimal refolding at intermediate (but stillincreased 30 compared to ambient) pressure levels. The depressurizationor interruptions may be 1 second, 2 seconds, 5 seconds, 10 seconds, 20seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 60minutes, 2 hours, 3 hours, 4 hours, and 5 hours.

Degassing is the removal of gases dissolved in solutions and is oftenadvantageous in the practice of the methods described herein. Gas ismuch more soluble in liquids at high pressure as compared to atmosphericpressure and, consequently, any gas headspace in a sample will be driveninto solution upon pressurization. The consequences are two-fold: theadditional oxygen in solution may chemically degrade the proteinproduct, and gas exiting solution upon repressurization may causeadditional aggregation. Thus, samples should be prepared with degassedsolutions and all headspace should be filled with liquid prior topressurization.

EXAMPLES Example 1 Use of High Pressure to Disaggregate and RefoldAggregates of Orencia® (CTLA-4Ig; abatacept)

Aggregation states of Orencia® (Orencia® is a registered trademark ofBristol-Myers Squibb Co., New York, N.Y., for pharmaceuticalpreparations for the treatment and prevention of auto-immune diseasessuch as rheumatoid arthritis), also known as abatacept or CTLA-4Ig, weremonitored in both commercial formulations and after exposure to pH 3 for3 hours. Orencia® is a soluble fusion protein, consisting of theextracellular domain of human cytotoxic T-lymphocyte-associated antigen4 (CTLA-4) fused to the modified Fc (hinge, CH2, and CH3 domains)portion of human immunoglobulin G1 (IgG1). Aggregates of CTLA-4Ig werepressure treated as a function of pH, pressure and additives asdescribed below.

For sample preparation, aggregates were generated in 10 mM phosphatebuffer at a pH of 3.0 at a protein concentration of 12 mg/ml. Thesamples were treated at these conditions until an aggregation extent of˜20% was achieved. The samples were then diluted to a concentration of0.5 mg/ml in the appropriate solution conditions. The samples wereplaced into sealed syringes and pressure treated. Atmospheric controlswere prepared under identical conditions and also stored in sealedsyringes.

Pressurization was increased at a rate of 500 bar/minute until thedesired pressure was achieved. During refolding, the temperature wasmaintained at 22 C (R.T.). The samples were held under pressure forapproximately 16 hours and then were depressurized at a rate of 500bar/five minutes. The samples were immediately prepared for SEC afterdepressurization.

Size Exclusion Analysis—High Pressure Liquid Chromatography (SEC)analysis of protein fractions was conducted on a Beckman Gold HPLCsystem (Beckman Coulter, Fullerton, Calif.) equipped with a TSK G3000SW_(XL) size exclusion column (Tosohaas). A filtered mobile phase of PBS(pH 7.2) at a rate of 1.0 ml/min was used, with an 10-25 ug proteinsample injection from a Beckman 507e autosampler. Absorbance wasmonitored at 215 nm.

Aggregation of CTLA-4Ig at low pH was achieved by diluting commercialformulations to a protein concentration of 12 mg/ml and treated at pH 3for 3 hours at 23° C. to induce aggregation. Two runs resulted in finalaggregate concentrations of 15 and 21 percent respectively (Labeled Init% Agg.—FIG. 1). Aggregate analysis was quantified by SEC. High pressuretreatment at pH 7 and higher resulted in ˜95% refolding yields andaggregate levels lower than commercial CTLA-4Ig preparations (Orencia®).

High pressure refolding of CTLA-4Ig as a function of pH is exemplifiedby aggregates (0.5 mg/ml) forming after exposure to pH 3, pressuretreatment at 2000 bar for sixteen hours at 25° C. and compared toatmospheric controls (FIG. 1). Buffer concentrations were 10 mM tomaintain a low ionic strength. Samples pressure treated in solutions ofpH 7 or higher resulted in ˜95% refolding yields, with a final aggregateconcentration of 0.8%. The final aggregate concentration of 0.8% afterpressure treatment is lower than what was present in the startingmaterial prior to pH 3 induced aggregate (1.2%).

Atmospheric controls treated at identical temperature and solutionconditions refolded to significantly lower levels relative to samplestreated at high pressure. The basis for the decreased refolding at lowerpHs is unknown. Error bars depict 95% confidence intervals. Throughoutthe entire analysis, total protein area was monitored to ensure thatprotein adsorption across the SEC column was minimal and the sizingmethod was quantifying aggregate levels accurately. Refolding at 5 mg/mlat pH 7.0 also resulted in refolding, with similar yields to the 0.5mg/ml samples (data not shown).

Studies were conducted to examine high pressure refolding of CTLA-4Igaggregates at pH 7.0 as a function of excipients; more specifically, toexamine the effect of excipients (250 mM arginine, 10% (w/v) sucrose,and 0.01% (w/v) Tween 20) on the refolding yield of CTLA-4Igacid-induced aggregates (0.5 mg/ml) at pH 7 (10 mM buffer) (FIG. 2).Excipients did not significantly increase the refolding yield oversamples that were refolded in buffer alone (low ionic strength solutionconditions) (FIG. 1). The presence of arginine decreased refoldingyields; this data is consistent with an ionic-strength dependent effect.It was previously observed that high pressure treatment in the presenceof 250 mM NaCl at pH 7 resulted in the aggregation of native CTLA-4Ig(data not shown).

To illustrate high pressure refolding of CTLA-4Ig aggregates at pH 7.0as a function of pressure, aggregates of CTLA-4Ig (0.5 mg/ml) weretreated at pH 7.0 (10 mM Buffer) as a function of pressure (0-3 kbar) at25° C. for 16 hours. Pressures of 2000 bar were sufficient for therefolding of the aggregate with yields of ˜95% relative to the initialaggregate concentration; increased pressures did not provide anyincreased yields (see FIG. 3).

Aggregation states of CTLA-4Ig were monitored in both commercialformulations and after exposure to pH 3 for 3 hours. Aggregates werepressure treated as a function of pH, pressure and additives. Thisillustrative study showed: high pressure refolded pH 3 inducedaggregates of CTLA-4Ig (0.5 mg/ml), reducing aggregate levels from 18%to 0.8% (95% yield); high pressure refolding reduced the aggregatelevels below those originally found in commercial formulations ofCTLA-4Ig (Orencia®) being 0.8% vs. 1.3%, respectively; existing nativeprotein is preserved during high pressure refolding in the appropriatesolution conditions, however high pressure treatment in solutions ofhigh ionic strength induced aggregation; refolding yields are notaffected by moderate differences of initial aggregate concentrations(21% vs. 15%); refolding yields are pH dependent below pH 7; and,refolding pressures of 2000 to 3000 bar produce equivalent results

Example 2 Use of High Pressure to Disaggregate and Refold Aggregates ofthe “Alliance mAb”

Aggregation states of a monoclonal antibody which was a gift of AllianceLaboratories were monitored after exposure to pH 3. The sequence andproperties of this aggregate were unknown. Aggregates of the monoclonalantibody were pressure treated at a pH of 7.2 as a function of pressureand additives, as described below.

Aggregate samples were generated by dialysis into buffer at a pH of 3.0at a protein concentration of 37 mg/ml. The samples were dialyzed in thebuffer for six hours and then maintained at room temperature for sixadditional hours. The samples were then diluted to a concentration ofeither 0.3 or 5 mg/ml in the appropriate solution conditions. Buffer andionic strength concentrations were 50 mM TES, pH 7.2, 150 mM NaCl. Thesamples were placed into sealed syringes and pressure treated.Atmospheric controls were prepared under identical conditions and alsostored in sealed syringes.

Pressure was increased at a rate of 500 bar/minute until the desiredpressure was achieved. During refolding, the temperature was maintainedat 22 degrees C. (room temperature). The samples were held underpressure for approximately 16 hours and then were depressurized at arate of 500 bar/five minutes. The samples were immediately prepared forSEC after depressurization.

Size Exclusion Analysis—High Pressure Liquid Chromatography(SEC)_analysis of protein fractions was conducted on a Beckman Gold HPLCsystem (Beckman Coulter, Fullerton, Calif.) equipped with a TSK G3000SW_(XL) size exclusion column (Tosohaas). A filtered mobile phase of PBS(pH 7.2) at a rate of 1.0 ml/min was used from a Beckman 507eautosampler. Absorbance was monitored at 215 nm.

Aggregation of the Alliance mAb at low pH was induced by diluting 50mg/ml formulations to a protein concentration of 33.5 mg/ml and exposureto pH 3 for 6 hours at 25° C. The final aggregate content was estimatedto be 32% percent. Aggregate analysis was quantified by SEC.

To illustrate high pressure refolding of Alliance mAb aggregates as afunction of pressure, aggregates of the Alliance mAb (5 mg/ml) formedafter exposure to pH 3 were pressure treated at 2000 bar for sixteenhours at 25° C. at a pH of 7.2. Pressures of 1 bar, 1000 bar, 1500 bar,2000 bar and 3000 bar were tested. Of the samples tested, onlyaggregates pressure treated at 1000 to 2000 bar resulted in increasedmonomer content (FIG. 4). Aggregates pressure treated to 3000 barresulted in increased aggregation, demonstrating the importance of therefolding window. Atmospheric controls treated at identical temperatureand solution conditions also had lower monomer content. High pressuretreatment at pressures of 1000-2000 bar resulted in the desiredrefolding.

Studies were conducted to examine high pressure refolding of AlliancemAb aggregates at pH7.2 as a function of excipients. In order to examinethe effect of excipients (250 mM arginine, 10% (w/v) sucrose, and 0.01%(w/v) Tween 80) on the refolding yield of Alliance mAb acid-inducedaggregates (5 mg/ml) at pH 7.2 (FIG. 5). Excipients did notsignificantly increase the refolding yield over samples that wererefolded in buffer alone, i.e., at pH 7.2 without aggregates present(FIG. 5).

To illustrate the high pressure refolding of the aggregates of theAlliance mAb, aggregates (0.3 mg/ml) were incubated at pH 7.2 as afunction of temperature (25-37 degrees C.) for 16 hours. Pressures of2000 bar at 25 degrees C. were sufficient for increasing the amount ofmonomer and increased temperature did not provide any increased yields.Increased temperature did not increase monomer peak area. (see FIG. 6).

Aggregation states of the Alliance mAb were monitored after exposure topH 3.0. Aggregates of the mAb were pressure treated at a pH of 7.2 as afunction of pressure and additives. This illustrative study showed: highpressure treatment of pH 3-induced aggregates at a pH of 7.2 resulted inincreased monomer peak area as analyzed by SEC in comparison toatmospheric conditions, having values of 1.6 million and 1.8 million,respectively; addition of additives such as 250 mM arginine, 0.05% Tween80, and 10% sucrose did not significantly effect the refolding yield ofthe Alliance mAb; elevated temperatures of 37° C. did not significantlyincrease the refolding yield of Alliance mAb aggregates at pressuretreated to 2000 bar at pH 7.2 at 25° C.; and, pressures of 3000 barinduced aggregation of native mAb. Ongoing analyticalultracentrifugation (AUC) studies in order to quantify the refoldingyields more accurately, as analysis of total peak area suggests thatsome protein adsorption may occur during SEC analysis.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

1. A method for refolding a sample of an antibody, comprising the stepsof: obtaining an antibody sample comprising a solution of antibodyexposed to low pH; adjusting the pH of the solution to above pH 5.0 ifnecessary; exposing the antibody sample to high hydrostatic pressure fora period of time; and reducing the hydrostatic pressure to atmosphericpressure; wherein the antibody sample after pressure exposure has ahigher content of monomeric antibody, a higher content of properlyfolded antibody, or a lower content of aggregated antibody than theantibody sample prior to the pressure exposure.
 2. The method of claim1, wherein the antibody is monoclonal.
 3. The method of claim 2, whereinthe content of monomeric or properly folded antibody is measured by amethod selected from a binding assay specific for the antibody,analytical ultracentrifugation, size exclusion chromatography, fieldflow fractionation, light scattering analysis, light obscurationanalysis, fluorescence spectroscopy, gel electrophoresis, GEMMA, nuclearmagnetic resonance spectroscopy, electron paramagnetic resonancespectroscopy, or reverse-phase chromatography.
 4. The method of claim 2,wherein the high hydrostatic pressure is between about 1500 bar and avalue about 50 bar below the denaturation pressure of the nativeantibody.
 5. The method of claim 4, wherein the high hydrostaticpressure is between about 1500 bar and about 3000 bar.
 6. The method ofclaim 2, wherein the ionic strength of the antibody sample comprising anantibody solution is less than a value about 25 mM below thedenaturation point of the native antibody.
 7. The method of claim 6,wherein the ionic strength of the antibody sample comprising an antibodysolution is less than about 200 mM.
 8. The method of claim 2, whereinthe antibody is abatacept.
 9. The method of claim 8, wherein the pH ofthe abatacept antibody solution is at or above about
 7. 10. The methodof claim 9, wherein sucrose is present in the antibody solution at aconcentration of about 5% to about 15%.
 11. The method of claim 10,wherein sucrose is present in the antibody solution at a concentrationof about 10%.
 12. The method of claim 7, wherein the ionic strength ofthe antibody solution is less than about 100 mM.
 13. The method of claim8, wherein the amount of aggregated antibody present in the antibodysample after pressure exposure is decreased by at least about 20% ascompared to the amount of aggregated antibody present in the antibodysample before pressure exposure.
 14. The method of claim 8, wherein theamount of aggregated antibody present in the antibody sample afterpressure exposure is less than about 1%.
 15. A preparation of abataceptantibody comprising less than about 1.0% aggregated antibody.
 16. Apreparation of abatacept antibody comprising about 0.8% or lessaggregated antibody.